This project is focused on the regulation and function of the MAPK Kinase Kinase MEKK3. MAPK signaling pathways have been shown to be essential for cell growth, differentiation and apoptosis. Given the critical contributions of MAPKs, it is important to elucidate the different MAPK signaling pathways and their targets in various biologic contexts, including healthy and diseased states. We originally cloned MEKK3 and determined that when overexpressed, it is capable of activating all four major MAPKs, namely ERK, JNK, p38 and ERK5, albeit to different extents. Overexpression of MEKK3 can also protect cells from some apoptotic insults and activate NF-kappaB. MEKK3 has been proposed to have a direct role in activating the IKK kinase complex in response to TNF and other stimuli. The IKK complex phosphorylates and thus causes degradation of the inhibitors of NF-kappaB, leading to activation of this transcription factor family. We have cloned an adaptor protein that binds to MEKK3 and links MEKK3 to the IKK kinase complex and to JNK activation. The gene for this adaptor protein has recently been identified as one of three loci mutated in patients with inherited Cerebral Cavernous Malformations (CCMs). CCMs are sporadically acquired or inherited vascular anomalies of the CNS characterized by clusters of dilated thin-walled blood vessels that predispose individuals to seizures and strokes. To explore the in vivo roles of the adaptor and of MEKK3 in health and in CCMs we have generated mice deficient in the adaptor and conditionally deficient in MEKK3. The homozygous loss of the adaptor results in early embryonic lethality, caused at least in part by defective angiogenesis. Initial experiments suggest a role for the adaptor in control of endothelial cell growth in culture. We are exploring the molecular mechanisms that may underlie the development of CCMs in patients. We have also begun to explore the role of MEKK3 in activation of MAPKs and NF-kappaB in select cell types in vivo with the use of conditionally deficient MEKK3 mice.